NMR Spectroscopic Identification of Urolithin G, a Novel Trihydroxy Urolithin Produced by Human Intestinal Enterocloster Species

Urolithins are gut microbiota metabolites of ellagic acid. Here, we have identified and chemically characterized a novel urolithin produced from urolithin D (3,4,8,9-tetrahydroxy urolithin) by in vitro incubation with different human gut Enterocloster species under anaerobic conditions. Urolithin G (3,4,8-trihydroxy urolithin) was identified by 1H NMR, 13C NMR, UV, HRMS, and 2D NMR. For the identification, NMR spectra of other known urolithins were also recorded and compared. Urolithin G was present in the feces of 12% of volunteers in an overweight-obese group after consuming an ellagitannin-rich pomegranate extract. The production of urolithin G required a bacterial 9-dehydroxylase activity and was not specific to the known human urolithin metabotypes A and B. The ability to produce urolithin G could be considered an additional metabolic feature for volunteer stratification and bioactivity studies. This is the first urolithin with a catechol group in ring A while having only one hydroxyl in ring B, a unique feature not found in human and animal samples so far.


■ INTRODUCTION
Ellagic acid and ellagitannins are polyphenols present in many food products, and they have been associated with biological effects that promote human health. 1 Ellagitannins are not absorbed in the small intestine 2 and are hydrolyzed by probiotic strains to release ellagic acid. 3 Ellagic acid is also poorly absorbed and reaches the gut in significant amounts. 2 In the gut, ellagic acid is converted by the gut microbiota into urolithins after lactone ring opening, decarboxylation, and sequential losses of hydroxyls to reach the final metabolites urolithin A (3,8dihydroxy urolithin) (Uro-A), isourolithin A (3,9-dihydroxy urolithin) (IsoUro-A), and (or) urolithin B (3-hydroxy urolithin) (Uro-B). 4,5 Other urolithin intermediates have been reported. 6 Urolithins are much better absorbed and are bioactive metabolites with effects on metabolic syndrome, diabetes, inflammation, cardiovascular, cognitive, and muscle functions. 7−10 The ability to produce different urolithins by Gordonibacter and Ellagibacter species is well established, 11,12 and the whole metabolic process to reach the final circulating metabolites was recently complemented with the new strain Enterocloster bolteae CEBAS S4A9 and representative strains of the closest relatives (E. bolteae DSM 29485, DSM 15670 T , Enterocloster asparagiformis DSM 15981 T , Enterocloster citroniae DSM 19261 T , Enterocloster clostidioformis DSM 933 T ). 13,14 The identification of the different urolithins produced in the gut is of interest as they can be responsible for the health effects observed after the intake of food containing ellagitannins and ellagic acid. Thus, pentahydroxy, tetrahydroxy, and trihydroxy urolithins were identified as intermediate metabolites produced before reaching the final urolithins mentioned above. 4−6 These intermediates have interest due to their potential biological effects in the gut and as intermediates in the biotechnological production of urolithins.
In the course of the elucidation of the metabolic pathways by which different bacterial strains produce urolithins, we tested the metabolism of urolithin D (3,4,8,9-tetrahydroxy urolithin) by different bacterial strains and discovered that most of the Enterocloster species tested yielded a potential novel urolithin which we called Urolithin G (Uro-G), which was not produced by Gordonibacter or Ellagibacter strains. 14  and other urolithin-producing bacteria (Gordonibacter urolithinfaciens DSM 27213 T and Ellagibacter isourolithinifaciens DSM 104140 T ) obtained from DSMZ culture collection were used to investigate their capacity to transform Uro-D as described recently. 13,14 Briefly, 2 mL of diluted inoculum were transferred to Wilkins-Chalgren anaerobe medium (WAM, Condalab, Madrid, Spain) (20 mL), obtaining an 1 H -13 C HSQC NMR (heteronuclear single quantum coherence) spectra were recorded using "hsqcetgpsi" pulse program (adiabaticpulsed version) with the gradient selected sequences with 256 transients and 1024 data points for each of 512 increments. The spectral widths were 4986 Hz (from 11.2 to 1.2 ppm) for 1 H and 23,892 Hz (from 187 to 7 ppm) for 13 C in HSQC experiments. The data were Fourier transformed into a 4 × 2 k matrix with appropriate apodization functions. The 1 JCH used was 145 Hz.
UHPLC-QTOF-MS−MS Analyses. Samples were also analyzed using an Agilent 1290 Infinity UPLC system coupled to a 6550 Accurate-Mass Quadrupole time-of-flight (QTOF) (Agilent Technologies, Waldbronn, Germany). This technique provided a better identification of the new compound based on its molecular formula (obtained using mass accuracy and isotopic pattern) and the MS/MS fragmentation pattern. The chromatographic and mass spectrometric conditions tested were those previously optimized for quantifying urolithins. 16 Briefly, separation was achieved on a reversed-phase Poroshell 120 EC-C18 column (3 × 100 mm, 2.7 μm) using the following mobile phases: water plus 0.1% formic acid (phase A) and ACN plus 0.1% formic acid (phase B) in a gradient mode. The flow rate was 0.5 mL/min, and the injection volume was 5 μL. Spectra were acquired in negative polarity with a m/z range of 100−1100. Besides, MS/MS parameters were optimized at a m/z range of 50−800 using a retention time window of 1 min, a collision energy from 20 to 40 V, and an acquisition rate of 4 spectra/s. Data were processed using the MassHunter Qualitative Analysis software (version B.10, Agilent Technologies, Waldbronn, Germany).
Analysis of Human Fecal Samples. The samples from the POMEcardio study (NTC01916239) were obtained, as reported previously. 17 In that study, a pomegranate extract was administered for 3 weeks to 49 overweight-obese volunteers (17 women and 32 men; BMI > 27 kg/m 2 ), and feces were collected at the end. As reported, the fecal samples were extracted and analyzed by UPLC-ESI-qTOF-MS and HPLC-DAD-SQ-MS. 15,16 The chromatograms were reanalyzed here to search for Uro-G and other new urolithins.
■ RESULTS AND DISCUSSION Urolithin D Conversion by Urolithin-Producing Bacteria. The HPLC-DAD-SQ-MS analyses showed that Uro-D was transformed by most of the Enterocloster strains tested, rendering the novel trihydroxy urolithin described below with a yield of 100%. Only two of the Enterocloster strains tested, E. bolteae DSM 15679 T and E. clostridioformis DSM 933 T , did not produce Uro-G (Table 1). In contrast, Gordonibacter urolithinfaciens DSM 27213 T and Ellagibacter isourolithinifaciens DSM 104140 T strains converted Uro-D into urolithin C (Uro-C, 3,8,9-trihydroxy urolithin), with a 54 and 100% yield, respectively (Table 1), which is a very distinctive feature. Only Uro-C, Uro-CR, 3,4,8-trihydroxy urolithin, and 3,4,9-trihydroxy urolithin could be produced by bacterial dehydroxylation of Uro-D ( Figure 1). The last two trihydroxyurolithins are new metabolites not previously identified as a product of gut microbes, and only one of them that we named Urolithin G (Uro-G) was produced after incubation of Uro-D with Enterocloster species (Figure 2).        (Table 2). To identify the new Uro-G, the 1 H NMR spectrum was recorded in AcN-d3 as this solvent is easily removed by reduced pressure concentration at low temperature (40°C), which allows the fast recovery of the isolated metabolite for further analyses and use as a standard and for biological assays. The spectrum in this solvent also showed the protons of the three phenolic hydroxyls ( Figure S1). However, the spectrum was not discriminant between the two possibilities ( Figure 1) since (i) the estimation obtained in the ChemDraw software was based on spectra recorded in DMSO-d6 (Table 3) and (ii) the solubility of the isolated urolithin in acetonitrile was not sufficient for some urolithins. Therefore, we also recorded the 1 H NMR spectra in DMSO-d6 (Table 3). The NMR spectra in both solvents were consistent with the spectra of a trihydroxy urolithin showing five aromatic H signals, 15 with those of other available urolithin standards (Supporting Figure  S1), and with the data reported in previous publications on NMR analysis of urolithins. 18−22 The 1 H NMR spectra in DMSO-d6 of Uro-D, Uro-A, and IsoUro-A were compared with the spectrum of Uro-G ( Figure  3). Uro-G showed very similar chemical shifts for H1 and H2 than Uro-D suggesting a similar hydroxylation pattern in ring A with hydroxyls at 3 and 4 positions. In addition, Uro-G showed almost identical chemical shifts for the H7, H9, and H10 as Uro-   A and very different from those of IsoUro-A, supporting a Uro-A hydroxylation pattern for the ring B.
The TOCSY experiment (Figure 4) clearly showed the coupling of H1 and H2, and H9 with H10, and the long-distance coupling of H-9 and H7. Uro-G does not have a singlet for H-7, confirming that this is not Uro-C or Uro-CR. In addition, Uro-G does not have a 1 H NMR signal at 7.02 ppm for H-8, which should be characteristic of the spectrum of the 3,4,9-trihydroxy urolithin isomer (ChemDraw estimation in DMSO-d6, Table  3).
The HSQC experiment also confirmed the Uro-G structure. In Figure 6, we show the HSQC results in which the five C−H carbons (DEPT) and the connected five protons of Uro-G are evidenced. The results also confirmed the 13 C NMR assignments ( Figure 5).
The UV spectrum of Uro-G ( Figure 7) with a BI/BII ratio of 0.29 also indicated the lack of hydroxyl at the 9-position of the urolithin nucleus, in contrast with the 9-hydroxy urolithins Uro-C (BI/BII 0.16) and Uro-CR (BI/BII 0.15) and in agreement with previous studies. 15 The other feasible isomer, 3,4,9trihydroxy urolithin, should have a UV spectrum with a BI/ BII ratio around 0.15, and thus, the UV confirmed the structure of Uro-G as 3,4,8-trihydroxy urolithin.
Uro-G was only obtained after Uro-D incubation with Enterocloster species that harbor 9-dehydroxylase activity. 14 In this study, we have only considered dehydroxylations of Uro-D by the dehydroxylase enzymes present in the bacteria used (Gordonibacter, Ellagibacter, and Enterocloster) (Figure 1). Other metabolites could have been considered to be produced from Uro-D by hydroxyl transfer as it has been reported for the conversion of pyrogallol (1,2,3-trihydroxy benzene) into phloroglucinol (1,3,5-trihydroxy benzene) by the anaerobic bacteria Pelobacter acidogallici 23 and Eubacterium oxidoreducens, 24 although this is very unlikely and has not been reported for the bacteria assayed in the present study.
For the first time, we have described the spectroscopic features of the new Uro-G produced from Uro-D in vitro. Therefore, the occurrence of this metabolite in human feces after the intake of ellagitannins would confirm the activity of human Enterocloster species in vivo. For this purpose, we revisited the analyses of human feces after the intake of pomegranate ellagitannins in the POMEcardio study. 17 This survey confirmed the occurrence of Uro-G in some of the fecal samples (6 out of 49 volunteers; 12%) ( Figure 8). However, Uro-G was a minor metabolite in the feces of volunteers belonging to both metabotypes A and B, and thus, its occurrence was not specifically associated with one of these urolithin-producing metabotypes.
This new urolithin could also be present in human biological fluids (plasma and urine) since some trihydroxy urolithin derivatives, such as Uro-C, have been detected in some cases. 25 However, this was not addressed in the present study.
To the best of our knowledge, Uro-G is the first urolithin with a catechol group in the A ring while having only one hydroxyl in the B ring, a unique feature not found in human and animal samples so far. ■ ASSOCIATED CONTENT